Analytical and clinical validation of diagnostic tests for the detection of leucospermia in beef bulls

The objectives of this study were to validate diagnostic tests to detect polymorphonuclear cells (PMNs) in bull semen, and to determine the prevalence of leucospermia in beef bulls with varying semen quality. We hypothesized that all tests have comparable diagnostic value, and that leucospermia is more prevalent in unsatisfactory breeders in association with poor semen quality. For the analytical validation, one ejaculate was obtained from five bulls. Aliquots of 50 × 10⁶ purified sperm were incubated in triplicate with six concentrations of purified bovine PMNs: 1) no PMNs, 2) 0.25 × 10⁶ PMN/ml, 3) 0.5 × 10⁶ PMN/ml, 4) 2.5 × 10⁶ PMN/ml, 5) 5 × 10⁶ PMN/ml, 6) 10 × 10⁶ PMN/ml. The PMNs were quantified using a hemacytometer, cytology, a leucocyte esterase dipstick test (LEDT), a peroxidase test, and CD45 immunolabeling. The number of leucocytes detected with the LEDT differed among treatments (P < 0.0001). The quantitative tests detected differences with the control treatment at a PMN concentration of ≥2.5 × 10⁶ PMN/ml (P < 0.0001). Sperm motion parameters after 4 h of incubation at 38 °C were lower in samples with ≥5 × 10⁶ PMN/ml (P < 0.05). For the clinical validation, semen samples from 305 beef bulls were evaluated. Unsatisfactory breeders (n = 83) had more CD45-positive cells (P = 0.016) and positive LEDT results (P = 0.008) than satisfactory breeders (n = 222). With CD45 immunostaining as the gold standard, the hemacytometer count had the highest clinical sensitivity (64.3 %) but the lowest specificity (73.3 %). A higher specificity was obtained with the peroxidase test (95.1 %) or semen cytology (98.8 %). In conclusion, the presence of ≥5 × 10⁶ PMN/ml was associated with decreased semen quality in beef bulls. The hemacytometer count was the most sensitive bull-side test. But due to the low specificity, positive hemacytometer counts should be confirmed with the identification of peroxidase-positive cells or morphological identification of leucocytes on semen cytology. The CD45 immunostaining is the gold standard for the diagnosis of leucospermia in bulls.